PACAP was first isolated from the hypothalami of sheep as a peptide promoting adenylate cyclase activity of the pituitary glands Biochemical Biophysical Research Communications, 164, 567-574 (1989)!. PACAP first isolated was one consisting of 38 amino acid residues. However, the presence of PACAP consisting of 27 residues on the N-terminal side was also revealed. Both are nearly equal in adenylate cyclase activating ability to each other. The former is referred to as PACAP38, and the latter is referred to as PACAP27. The expression of a PACAP having the same structure as that of sheep was also proved in humans, which suggested that PACAPs are important peptides conserved beyond species Biochem. Biophys. Res. Commun., 166, 81-89 (1990)!. For the distribution thereof in organs, they are observed not only in the brain hypothalami, but also in the pituitary glands, the testes and the adrenals (Endocrinology, 129, 2787-2789). At present, PACAPs such as PACAP27 to PACAP38 (U.S. Pat. No. 5,128,242) and PACAP23 to PACAP26 (European Patent Publication No. 0467279A3) have been reported.
Physiological actions of PACAPs diversely varies according to their occurrence sites. Various actions of the PACAPs as described below have hitherto been reported:
(1) Promotion of cAMP production in primary culture cells of the rat pituitary glands A. Miyata et al., Biochem. Biophys. Res. Commun., 164, 567-574 (1989)!;
(2) Promotion of secretion of GH, ACTH, PRL and LH in the rat pituitary gland superfusion process A. Miyata et al., Biochem. Biophys. Res. Commun., 164, 567-574 (1989)!;
(3) Production of cAMP in adrenomedullary chromaffinoma-derived cells PC12h and promotion of neurite outgrowth T. Watanabe et al., Biochem. Biophys. Res. Commun., 173, 252-258 (1990), and K. Okazaki et al., FEBS Letters, 298, 49-56 (1992)!;
(4) Promotion of interleukin-6 production in pituitary gland culture cells I. Tatsuno et al., Endocrinology, 129, 1797-1804 (1991)!; and
(5) Promotion of cAMP production in primary culture of rat astrocytes and promotion of action preventing nerve cell death Biochem. Biophys. Res. Commun., 168, 1027-1033 (1990)!.
In order for PACAP to exhibit its action, the presence of a receptor specific for PACAP in target organs and cells is indispensable.
Receptor binding experiments using radioactive iodine-labeled PACAP27 (.sup.125 I! PACAP27) have proved the presence of a PACAP receptor. Namely, when a membrane fraction prepared from a tissue is mixed with .sup.125 I ! PACAP27 and reacted for an appropriate period of time, binding of .sup.125 I! PACAP27 to the membrane fraction is observed. This binding is inhibited by unlabeled PACAP27 or PACAP38, but not inhibited by VIP, an analogous peptide of the PACAPs. This result suggests that a substance specifically binding to the PACAPs occurs in the tissue. Such binding activity is highest in membrane fractions of the brain hypothalami, and also observed in the pituitary glands, the adrenals and the like Endocrinology, 127, 272-277 (1990)!. Further, a body of PACAP binding activity observed in membrana cerebri fractions, namely a receptor, is deduced to be a protein having a molecular weight of 57,000 from a technique (so-called affinity-label experiment) comprising binding .sup.125 I! PACAP27 to the membrana cerebri fraction, crosslinking .sup.125 I! PACAP27 and the body of its binding activity with a crosslinking reagent, then subjecting the product to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, and analyzing by autoradiography Biochem. Biophys. Res. Commun., 171, 838-844 (1990)!.
It is expected that clarification of some fundamental properties of this specific receptor allows elucidation of additional various properties of the PACAP receptor to proceed more than before. In particular, cloning of cDNA coding for the receptor protein and structure analysis thereof enable elucidation of the mechanism of its mutual interaction with a ligand, production of receptor-agonists and antagonists and detailed analysis of sites of action by in situ hybridization using said cDNA. Although cloning of the VIP, secretin and growth hormone releasing factor receptor proteins cDNA has been reported, the cloning of the PACAP receptor has not. These three kinds of bioactive peptides have also showed a capital similarity in the structures of their receptor proteins. For the PACAP receptors, however, cloning of cDNA has hitherto not been carried out.
Recently, the following five documents reported amino acid sequences for a rat PACAP receptor protein and nucleotide sequences of DNAs coding for the protein Document 1: Biochemical and Biophysical Research Communication, 194, 1, pp.133-143, 1993; Document 2: Federation of European Biochemical Societies (FEBS), 329, 1 and 2, pp. 99-105; Document 3: Proceedings of the National Academy of Science, USA, 90, pp. 6345-6349, 1993; Document 4: Nature, 365, pp. 170-175, 1993 and Document 5: Neuron, 11, pp.333-342, 1993). Among them, the amino acid sequences and the nucleotide sequences described in the Documents 1, 2, 4 and 5 are identified with the amino acid sequence for a rat PACAP receptor protein and with the nucleotide sequence for a DNA coding for the protein. The amino acid sequence described in Document 3 is different from the amino acid sequence of the present invention for a rat PACAP receptor protein in one amino acid, and the nucleotide sequence of Document 3 is also different from the nucleotide sequence of the present invention in one nucleotide. All of the five documents were published after Jun. 24, 1993 which is one of the priority dates of the present invention.
In general, when specific binding substances such as receptors are purified, affinity column chromatography applying its mutual interaction with the specific binding substance (for example, ligands for receptors) are frequently used. A process using an affinity column in which a ligand is fixed on a carrier is simplest. However, many successful examples of complicated affinity chromatography are known in which the specific mutual interaction between avidin and biotin is utilized for purification of receptors. This process comprises synthesizing a biotinylated ligand in which biotin is bound to an appropriate site, and specifically capturing a receptor on a carrier on which avidin is fixed through the biotinylated ligand Methods in Enzymology, 184, 244-274 (1990)!. This process suffers from the problem of designing the biotinylated ligand having affinity for both the receptor and avidin, and examination is required in purifying PACAP receptor.
PACAP38 and PACAP27 are peptides represented by the following amino acid sequences, respectively:
__________________________________________________________________________ PACAP38 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr 1 5 10 Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val 15 20 25 Leu Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn Lys-NH.sub.2 30 35 (SEQ ID NO: 46-NH.sub.2) PACAP27 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr 1 5 10 Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val 15 20 25 Leu-NH.sub.2 (SEQ ID NO: 47-NH.sub.2) __________________________________________________________________________